Effects of Thermal Stress on Hematological And

AgroLife Scientific Journal - Volume 7, Number 1, 2018 The obtained orthophotomap can be used as Calina A., Calina J., Milut M., 2015. Study on Levelling ISSN 2285-5718; ISSN CD-ROM 2285-5726; ISSN ONLINE 2286-0126; ISSN-L 2285-5718 raster graphic support for any future project Works Made for Drawing Tridimensional Models of Surface and Calculus of the Volume of Earthwork. 4th requiring geographic information, being International Conference on Agriculture for Life, Life EFFECTS OF THERMAL STRESS ON HEMATOLOGICAL generated at a resolution of 2.33 cm/pixel, for Agriculture, Book series:Agriculture and AND METABOLIC PROFILES IN BROWN BULLHEAD, allowing easy identification of the desired Agricultural Science Procedia, 6, p. 413-420. Ameiurus nebulosus (LESUEUR, 1819) details while also being useful as teaching Calina J., Calina A., Babuca N.I., 2014. Study on the material in specialized disciplines (for implementation of GIS databases in achieving the 1 1 1 1 1 general urban plan. 14th International Daniel COCAN , Florentina POPESCU , Călin LAŢIU , Paul UIUIU , Aurelia COROIAN , 1 1 1 2 georeferencing, vectoring etc.). Multidisciplinary Scientific GeoConference SGEM, Camelia RĂDUCU , Cristian O. COROIAN , Vioara MIREŞAN , Antonis KOKKINAKIS , Furthermore, raw images can become input Book 2, Vol. 2, p. 817-824. Radu CONSTANTINESCU1 data for other graphics processing projects, and Dimitru G., 2007. Sisteme Informatice Geografice by anonymizing the information stored in the Geographic Information Systems (GIS). Blue 1University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, Faculty of Animal Publishing House, Cluj-Napoca, Romania. database, the geographic information system Science and Biotechnologies, 3-5 Mănăştur Street, Cluj-Napoca 400372, Romania Goodchild M.F.,2009. Geographic information systems 2 can be a practical application (laboratory) and science: today and tomorrow. Annals of GIS,15 Aristotle University of Thessaloniki, School of Forestry and Natural Environment, Laboratory of support for the students of the "Land and (1), p. 3-9, doi: 10.1080/19475680903250715. Wildlife and Freshwater Fisheries, P.O. Box. 241, 54124 Thessaloniki, Greece Cadaster Measurement". Malczewski J., 2009. GIS based multicriteria decision The geographically designed information analysis: a survey of the literature. International Corresponding author: [email protected] system can be considered a pilot project for the Journal of Geographical Information Science, 20 (7), p. 703-726. doi: 10.1080/13658810600661508. development of other GISs, thus avoiding Abstract Negula I. D., Badea A., Moise C., Poenaru V., 2017. possible future mistakes, correct estimation of Earth Observation Satellite Data in Support of Water Physiological stress response of Brown bullhead (Ameiurus nebulosus) was studied under laboratory conditions. This needs, adaptation, optimization and reuse of Management for Agriculture. AgroLife Scientific species is well known for the resistance and adaptability to environmental factors. In this sense, metabolic and forms, techniques and working methods, etc. Journal 6 (2), 133-136 hematologic profiles were determined for low (6o C), medium (18o C) and high (31o C) water temperatures. An increase Olinic T., Olinic E., 2016. The Effect of Quicklime in blood cell numbers (RBC, WBC) and hematocrit (Ht) was determined under high temperature conditions, as a Stabilization on Soil Properties. 5th International REFERENCES response to low dissolved oxygen. Platelet count (PLT) remained relatively constant regardless of the water Conference on Agriculture for Life, Life for temperature. The metabolic profiles showed significant and very significant differences for most of the analyzed indices.

Agriculture, Book series: Agriculture and The differences were due to variations in temperature that influenced appetite and ingestion of feed, respectivelly the Badea A.C., Badea G., David V., 2015. Aspects Agricultural Science Procedia 10, p. 444-451. respiratory function by vasoconstriction or hypoxia. about Green Management of Urban Areas in Sui D., 2014. Opportunities and Impediments for Open Romania. 15th International Multidisciplinary GIS. Transactions in GIS, 18 (1), p. 1-24, Key words: Ictaluridae, invasive species, blood analysis, medial parameters, adaptability. Scientific GeoConference SGEM, Book 2, Vol. 2, p. doi:10.1111/tgis.12075. 721-728 Wadsworth R., Treweek J., 1999. Geographical conditions (Kapusta et al., 2010; Popescu, Băduț M., 2007. GIS – Geographic Information Systems, Information Systems for Ecology. Harlow: Longman. INTRODUCTION Practical Foundations. Sisteme Informatice ***http://www.earthexplorer.com/200802/exploration_a 2014), so it can withstand low dissolved oxygen Geografice, fundamente practice, (Editura) Blue, nd_gis_closing_the_productivity_gap.asp. Brown bullhead (Ameiurus nebulosus) is part of values, low water pH (Frank, 2015) and large Publishing House, Cluj-Napoca, Romania. ***http://www.esri.com. the Ictaluridae family. It is native to North temperature variations (Scott and Crossman, Burghilă C., Bordun C., Cîmpeanu S.M., Burghilă D., ***http://lib.icimod.org/record/21429/files/attachment_8 Badea A., 2016. Why mapping ecosystems services America and has its origin area between Hudson 1973). All these physiological features 5.pdf . Bay, St. Lawrence - Great Lakes, the American correlated with specific anatomical structures is a must in EU biodiversity strategy for 2020? ***https://www.nationalgeographic.org/encyclopedia/ge AgroLife Scientific Journal, 5(2), p. 28-37. ographic-information-system-gis/. Atlantic limit and the Mississippi River (Craig et (well-developed olfactory and gustative Burrough P.A., 2007. Development of intelligent ***http://www.opengis.unibuc.ro. al., 2015). It was introduced in various parts of analyzers, venomous glands, robust body geographical information systems. International Europe (Holčik, 1991), Asia and the Pacific formations) (Moldowan et al., 2015; Popescu et Journal of Geographical Information Systems, 6, Islands, including New Zealand (Collier et al., al., 2015) contribute to a very good adaptation in p. 1-11. -th 2016) at the end of the XIX century (1885). The the new habitats, becoming a dominant species data regarding the occurrence of this species in with major influences on endemic fish (Lenhardt Romania is controversial. Vasiliu (1959) et al., 2010). Brown bullhead is usually found mentioned its artificial introduction at the in benthic zones, were it competes for food and beginning of the XX-th century (1908). Bănărescu resources with various demersal species (1964) mentioned its migration from western (Kapusta et al., 2010). When found in natural European countries and noticed its presence in water bodies, a high survival rate is noticed the Romanian Danube sector, Baziaş area, around because of its territorial and parental-protective 1940. It was introduced for diversity purposes behaviour. The feeding spectrum is represented like sport fishing in these new habitats. It also by chironomidae larvae (Kline and Wood, 2011; became the subject of aquaculture (Dunham, Frank, 2015), diptera, fish eggs, fish juveniles 2006; Marković et al., 2012) and artificial and crustaceans such as crayfish (Keast, 1985; reproduction (Fobes, 2013). Raney and Webster, 2011). All the above are The Brown bullhead is recognized for its reasons for which the Brown bullhead is adaptability to difficult environmental considered a harmful species. Measures are 33 currently taken to limit its spread in both each group. The samples were transferred into (λ = 340 nm) (UV); CREAT-creatine (λ = 510 increase of erythrocyte counts. In response to artificial and natural environments (Danalache et 95 IU (4 mL) Li-Heparin anticoagulant nm) (NIS)], lipid profile [CHOL-cholesterol (λ environmental conditions, fish specific al., 2017). vacutainers and transported under refrigeration = 500 nm) (NIS); TGC-triglyceride (λ = 550 hematopoiesis in the kidneys and spleen In the case of aquatic organisms, temperature conditions (+ 2°C) to the hematology nm) (EP)], carbohydrate profile [BG-blood (Maekawa and Kato, 2015) is accelerated and a along with other physical and chemical factors laboratory of UASVM Cluj-Napoca. The glucose (λ = 50 nm) (NIS)], mineral profile higher number of erythrocytes are able to carry play an essential role in the progress of following determinations were performed: [TC-total calcium (λ = 575 nm) (EP); P - more efficiently the reduced amount of physiological processes expressed by growth HGB (hemoglobin), HCT (hematocrit), MCV phosphorus (λ = 340 nm) (UV); Fe-blood iron dissolved oxygen in the water. and intensity of metabolism (Peck et al., 2005). (mean corpuscular volume), MCH (mean (λ = 630 nm) (EP); Na -sodium (λ = 405 nm) The hemoglobin (Hg) varies from 5.5±0.196 Each species has optimal temperature range, in corpuscular hemoglobin), MCHC (mean (NIS); K -potassium (λ = 380 nm) (UV)], g/dL (at 18°C), to 7.66±0.638 g/dL (at 31°C) which these physiological processes run corpuscular hemoglobin concentration), RBC enzyme profile [ALP-alkaline phosphatase (λ = and 10.2±0.388 g/dL (at 6 °C). Fish have properly. If these conditions are not provided, a (red blood cell count) WBC (white blood cell 405 nm) (CR); GGT-gamma-glutamyl specific anatomical and physiological stressful state appears and has corresponding count), PLT (platelets) and the leukocyte transpeptidase; AST -aspartate adaptations that coordinate and adjust the consequences: decrease in metabolic rate, slow formula. The hematocrit was determined by aminotransferase; ALT -alanine amount of hemoglobin in the blood flow. This growth rate and installation of pathologic state centrifugation at 12.000 rpm/3 min. aminotransferase; AM EP-serum amylase; mechanism is based on the variation of medial (Barton, 2002). Hemoglobin concentration and erythrocytes LDH-lactate dehydrogenase (λ = 340 nm) conditions and the chemical modulation Blood is the most efficient stress indicator count were determined by spectrophotometry (CR); LIP -lipase (λ = 580 nm) (CR); CPK - stimulus. Both act on the oxygen requirements (Hattingh, 1977; Marin et al., 2015; Simide et techniques (UV-VIS Screen Master Touch). creatine phosphokinase (λ = 340 nm) (CR); TB of different tissues (de Souza and Bonilla- al., 2016). Biochemical and hematological studies End-Point colorimetric reaction was used for -total bilirubin]. Oxidative stress was also Rodriguez, 2007). Temperature and dissolved on fish are usually made for high economical hemoglobin determination. Readings were determined by the antioxidant SOD-superoxide oxygen levels from water are usually found in value species. In general, Brown bullhead studies performed in the NIS range spectra using as dismutase enzyme (λ = 505 nm). The statistical inverse relationship. Tissular oxygen demand is on hematological and biochemical properties were reagent 0.4% ammonium hydroxide NH4OH interpretation of the obtained data from no longer provided under hyperthermia sporadic and made the subject of cytological and in distilled water. Turbidimetric and biochemical analyzes was performed with the conditions. The physiological response to this pathophysiological researches (Rowan, 2007; colorimetric EP-type reaction with visible GraphPad Prism 6 software, using ANOVA thermal stress factor is manifested by variations Baumann et al., 2008). Despite the fact that the reading was used for the determination of test for multiple comparisons. Tables and of cortisol and blood glucose (Martínez-Porchas Brown bullhead is considered an invasive and erythrocytes using as a reagent acetic solution charts were designed in Word and Excel, et al., 2009) witch lead to blood pH alterations. undesirable species in Romania, studies regarding of sodium sulfate Na2SO4 in distilled water Microsoft Office 2010. Hemoglobin is highly sensitive to pH changes, its physiological traits have not been carried out. (Gowen's reagent). The leukocyte formula was Bohr reduction of Hg-O2 affinity) and Root Our study shows the expression of thermal stress obtained by microscopic reading of the RESULTS AND DISCUSSIONS (decrease in carrying capacity) effects occur in the blood constituents of the Brown bullhead. obtained blood smears. Coloration was under stress conditions (Rummer and Brauner, The obtained results give fundamental arguments performed by Reag-Quick-Panoptic [Reag-Fix Hematological analyzes show a numerical 2015). In Table 1 high levels of blood glucose in understanding the process of adaptation by Panoptic (fixative) - Reag-Red Panoptic increase in blood figurative elements, (BG) are presented, both hypo- and physiological mechanisms tested at extreme (eosinophil) - Reag-Blue Panoptic (basophil)] proportionally to the increase of temperature. hyperthermia leading to increased hemoglobin temperatures. and cleaning with bidistilled water. RBC For leukocytes, at 6oC the mean value was when thermal stress is installed. (MCV, MCH, and MCHC) was calculated 16.35 ± 1.596 WBC x 103/μL, at 18°C the The results of our study indicate that the highest MATERIALS AND METHODS based on the formulas reported by Ghergariu mean value was 17.20 ± 0.536 WBC x 103/μL hemoglobin concentration occurs for low water et al. (1999): (d = 0.85**, P <0.01) and at 31oC the mean temperatures (LT). Collateral and cumulative The Brown bullhead specimens from this study Mean Corpuscular Volume value was 18.12 ± 0.529 x 103/μL. physiological adaptations may be the response were sampled from Stejeriș Lake, Cluj County. The lowest mean value for erythrocytes was to environmental factors as previously Three groups of 50 specimens were constituted. obtained at 18oC (RBC = 0.39 ± 0.016 x described. Increased hemoglobin levels occur in The average body weight (BW) was 88.64±2.18 Mean Corpuscular Hemoglobin 106/μL) and the maximum mean value was hypothermia because of vasoconstriction g and the average total length (TL) was obtained at 31oC (RBC = 0.88 ± 0.11 x (especially in skeletal muscle capillaries) 19.85±0.14 cm. Each group was placed in 500L 106/μL). Intermediate mean value was obtained correlated with significant increase in water tanks for 21 days at three different Mean Corpuscular Hemoglobin Concentration at 6oC (RBC = 0.50 ± 0.032 x 106/μL). mitochondrial density (Johnson and Dunn, temperatures [Group 1: 6oC - low temperature Correlations between blood cells counts (WBC 1987). Blood circulation is carried out at a low (LT); Group 2: 18oC - normal temperature (NT); and RBC) and temperature were mentioned by rate, especially at the peripheral level and the o Group 3: 31 C - high temperature (HT)]. The biochemical profile obtained by Bozorgnia et al. (2011) and Witeska (2013). demand for tissular oxygen is no longer assured. In order to observe the physiological status of spectrophotometry techniques with (λ) The increase of figurative blood count is a A higher concentration of hemoglobin is Brown bullhead and the influence of thermal wavelength reading includes: protein profiles stress state indicator. necessary in this case. stress on the hematological profile, blood [TP -total protein (λ = 546 nm) (EP); ALB - The physiological response of fish regarding By comparing our hypothermic stress generating samples were collected by caudal vein puncture albumin (λ = 630 nm) (VIS); GLOB -globulin high water temperatures (when dissolved experiment to the conditions of the Arctic from 10 randomly selected specimens from (λ = 405 nm) (EP); BUN -blood urea nitrogen oxygen level is low) is expressed by the environment, some adaptive differences can be

34 currently taken to limit its spread in both each group. The samples were transferred into (λ = 340 nm) (UV); CREAT-creatine (λ = 510 increase of erythrocyte counts. In response to artificial and natural environments (Danalache et 95 IU (4 mL) Li-Heparin anticoagulant nm) (NIS)], lipid profile [CHOL-cholesterol (λ environmental conditions, fish specific al., 2017). vacutainers and transported under refrigeration = 500 nm) (NIS); TGC-triglyceride (λ = 550 hematopoiesis in the kidneys and spleen In the case of aquatic organisms, temperature conditions (+ 2°C) to the hematology nm) (EP)], carbohydrate profile [BG-blood (Maekawa and Kato, 2015) is accelerated and a along with other physical and chemical factors laboratory of UASVM Cluj-Napoca. The glucose (λ = 50 nm) (NIS)], mineral profile higher number of erythrocytes are able to carry play an essential role in the progress of following determinations were performed: [TC-total calcium (λ = 575 nm) (EP); P - more efficiently the reduced amount of physiological processes expressed by growth HGB (hemoglobin), HCT (hematocrit), MCV phosphorus (λ = 340 nm) (UV); Fe-blood iron dissolved oxygen in the water. and intensity of metabolism (Peck et al., 2005). (mean corpuscular volume), MCH (mean (λ = 630 nm) (EP); Na -sodium (λ = 405 nm) The hemoglobin (Hg) varies from 5.5±0.196 Each species has optimal temperature range, in corpuscular hemoglobin), MCHC (mean (NIS); K -potassium (λ = 380 nm) (UV)], g/dL (at 18°C), to 7.66±0.638 g/dL (at 31°C) which these physiological processes run corpuscular hemoglobin concentration), RBC enzyme profile [ALP-alkaline phosphatase (λ = and 10.2±0.388 g/dL (at 6 °C). Fish have properly. If these conditions are not provided, a (red blood cell count) WBC (white blood cell 405 nm) (CR); GGT-gamma-glutamyl specific anatomical and physiological stressful state appears and has corresponding count), PLT (platelets) and the leukocyte transpeptidase; AST -aspartate adaptations that coordinate and adjust the consequences: decrease in metabolic rate, slow formula. The hematocrit was determined by aminotransferase; ALT -alanine amount of hemoglobin in the blood flow. This growth rate and installation of pathologic state centrifugation at 12.000 rpm/3 min. aminotransferase; AM EP-serum amylase; mechanism is based on the variation of medial (Barton, 2002). Hemoglobin concentration and erythrocytes LDH-lactate dehydrogenase (λ = 340 nm) conditions and the chemical modulation Blood is the most efficient stress indicator count were determined by spectrophotometry (CR); LIP -lipase (λ = 580 nm) (CR); CPK - stimulus. Both act on the oxygen requirements (Hattingh, 1977; Marin et al., 2015; Simide et techniques (UV-VIS Screen Master Touch). creatine phosphokinase (λ = 340 nm) (CR); TB of different tissues (de Souza and Bonilla- al., 2016). Biochemical and hematological studies End-Point colorimetric reaction was used for -total bilirubin]. Oxidative stress was also Rodriguez, 2007). Temperature and dissolved on fish are usually made for high economical hemoglobin determination. Readings were determined by the antioxidant SOD-superoxide oxygen levels from water are usually found in value species. In general, Brown bullhead studies performed in the NIS range spectra using as dismutase enzyme (λ = 505 nm). The statistical inverse relationship. Tissular oxygen demand is on hematological and biochemical properties were reagent 0.4% ammonium hydroxide NH4OH interpretation of the obtained data from no longer provided under hyperthermia sporadic and made the subject of cytological and in distilled water. Turbidimetric and biochemical analyzes was performed with the conditions. The physiological response to this pathophysiological researches (Rowan, 2007; colorimetric EP-type reaction with visible GraphPad Prism 6 software, using ANOVA thermal stress factor is manifested by variations Baumann et al., 2008). Despite the fact that the reading was used for the determination of test for multiple comparisons. Tables and of cortisol and blood glucose (Martínez-Porchas Brown bullhead is considered an invasive and erythrocytes using as a reagent acetic solution charts were designed in Word and Excel, et al., 2009) witch lead to blood pH alterations. undesirable species in Romania, studies regarding of sodium sulfate Na2SO4 in distilled water Microsoft Office 2010. Hemoglobin is highly sensitive to pH changes, its physiological traits have not been carried out. (Gowen's reagent). The leukocyte formula was Bohr reduction of Hg-O2 affinity) and Root Our study shows the expression of thermal stress obtained by microscopic reading of the RESULTS AND DISCUSSIONS (decrease in carrying capacity) effects occur in the blood constituents of the Brown bullhead. obtained blood smears. Coloration was under stress conditions (Rummer and Brauner, The obtained results give fundamental arguments performed by Reag-Quick-Panoptic [Reag-Fix Hematological analyzes show a numerical 2015). In Table 1 high levels of blood glucose in understanding the process of adaptation by Panoptic (fixative) - Reag-Red Panoptic increase in blood figurative elements, (BG) are presented, both hypo- and physiological mechanisms tested at extreme (eosinophil) - Reag-Blue Panoptic (basophil)] proportionally to the increase of temperature. hyperthermia leading to increased hemoglobin temperatures. and cleaning with bidistilled water. RBC For leukocytes, at 6oC the mean value was when thermal stress is installed. (MCV, MCH, and MCHC) was calculated 16.35 ± 1.596 WBC x 103/μL, at 18°C the The results of our study indicate that the highest MATERIALS AND METHODS based on the formulas reported by Ghergariu mean value was 17.20 ± 0.536 WBC x 103/μL hemoglobin concentration occurs for low water et al. (1999): (d = 0.85**, P <0.01) and at 31oC the mean temperatures (LT). Collateral and cumulative The Brown bullhead specimens from this study Mean Corpuscular Volume value was 18.12 ± 0.529 x 103/μL. physiological adaptations may be the response were sampled from Stejeriș Lake, Cluj County. The lowest mean value for erythrocytes was to environmental factors as previously Three groups of 50 specimens were constituted. obtained at 18oC (RBC = 0.39 ± 0.016 x described. Increased hemoglobin levels occur in The average body weight (BW) was 88.64±2.18 Mean Corpuscular Hemoglobin 106/μL) and the maximum mean value was hypothermia because of vasoconstriction g and the average total length (TL) was obtained at 31oC (RBC = 0.88 ± 0.11 x (especially in skeletal muscle capillaries) 19.85±0.14 cm. Each group was placed in 500L 106/μL). Intermediate mean value was obtained correlated with significant increase in water tanks for 21 days at three different Mean Corpuscular Hemoglobin Concentration at 6oC (RBC = 0.50 ± 0.032 x 106/μL). mitochondrial density (Johnson and Dunn, temperatures [Group 1: 6oC - low temperature Correlations between blood cells counts (WBC 1987). Blood circulation is carried out at a low (LT); Group 2: 18oC - normal temperature (NT); and RBC) and temperature were mentioned by rate, especially at the peripheral level and the o Group 3: 31 C - high temperature (HT)]. The biochemical profile obtained by Bozorgnia et al. (2011) and Witeska (2013). demand for tissular oxygen is no longer assured. In order to observe the physiological status of spectrophotometry techniques with (λ) The increase of figurative blood count is a A higher concentration of hemoglobin is Brown bullhead and the influence of thermal wavelength reading includes: protein profiles stress state indicator. necessary in this case. stress on the hematological profile, blood [TP -total protein (λ = 546 nm) (EP); ALB - The physiological response of fish regarding By comparing our hypothermic stress generating samples were collected by caudal vein puncture albumin (λ = 630 nm) (VIS); GLOB -globulin high water temperatures (when dissolved experiment to the conditions of the Arctic from 10 randomly selected specimens from (λ = 405 nm) (EP); BUN -blood urea nitrogen oxygen level is low) is expressed by the environment, some adaptive differences can be

35 observed. Fish from Arctic regions have low oxide (NO) that acts as a vasodilator (Sidell and volume of erythrocytes also increases (Gillooly gradual and directly proportional decrease of hemoglobin levels (directly correlated with O'Brien, 2006). Brown bullhead is not native to and Zenil-Ferguson, 2014), directly affecting mean corpuscular volume (MCV) values. It is myoglobin) (Wells et al., 1980). This is a cold areas, thus lacking this phylogenetic the hematocrit value. Hematopoiesis is also possible to observe a higher value of this phylogenetic adaptability of fish species living in adaptation. accelerated in cases of hyperthermia as a result index at low temperatures (6°C MCV = cold waters, which have a high level of nitric of decreasing the erythrocyte-carrying capacity 76.33±5.779 fl), respectively a lower value in (Root effect), resulting the increase of case of high temperatures (31°C MCV = A B hematocrit. The mechanism consists in 38.66±2.109 fl). The average value of mean activating circulating stress hormones corpuscular volume recorded at 18 oC (MCV = (catecholamine) in hyperthermia and implicitly 60.90±1.077 fl) can be found between the in hypoxia (Lai et al., 2006), which accelerate highest and lowest values. hematopoietic processes. Mean corpuscular hemoglobin showed the Platelets (PLT) represent the only lowest value under control conditions (18oC hematological parameter that does not show MCH = 11.1±0.345 pg), value close to that any variation regardless of temperature resulting from hyperthermia (31oC MCH = 3 o intensity (PLT = 20.55±0.402 x 10 /μL - 6 C; 11.56±1.522 pg). Under hypothermia, the mean PLT = 20.30±0.434 x 103/μL - 18oC; PLT = corpuscular hemoglobin was higher (6oC MCH C D 3 20.30±0.636 x 10 /ΜL - 31 °C). = 17.86±1.868 pg). The erythrocyte count variation, as can be Mean corpuscular hemoglobin concentration seen in Figure 1, also led to the change of the showed the following mean values: 6°C - erythrocyte indices. Thus, the gradual increase MCHC = 24.30±2.503 g/dL; 18°C - MCHC = in the number of erythrocytes is correlated 31.30±0.454 g/dL; 31°C - MCHC = (according to the calculation formula) with a 22.57±1.176 g/dL.

E F

G H

Figure 1. Mean values and standard deviation of number of erythrocytes (A), number of leukocytes (B), hematocrit Figure 2. Leukocyte formula (%) in Brown bullhead (Ameiurus nebulosus) (C), hemoglobin content (D), mean corpuscular volume (E), mean corpuscular hemoglobin (F), mean concentration under different temperature conditions of corpuscular hemoglobin (G) and platelets (H) of Brown bullhead at the control (normal) temperature (CT), high temperature (HT) and lower temperature (LT) Figure 2 graphically represents the leukocyte shows at 18oC the following values of the formula of brown bullhead subjected to thermal leucocyte formula: NEU = 35%; EOS = 20%; The increase in hematocrit is gradually made 31oC - Ht = 27.55±1.461%. Previous studies stress. Leukocytes are reported as percentage LYM = 30%; MONO = 10%; BAS = 5%. At and directly proportional to the temperature. regarding blood analyzes of fish and other (relative values) and their values were obtained 6oC, the leukocyte formula shows the following The obtained values are as follows: 6oC - Ht = vertebrates have shown that when from smear readings. The graphic representation values: NEU = 7%; EOS = 3%; LYM = 80%; 18.38±0.443%; 18°C - Ht = 22.30±0.317%; environmental temperature increases, the

36 observed. Fish from Arctic regions have low oxide (NO) that acts as a vasodilator (Sidell and volume of erythrocytes also increases (Gillooly gradual and directly proportional decrease of hemoglobin levels (directly correlated with O'Brien, 2006). Brown bullhead is not native to and Zenil-Ferguson, 2014), directly affecting mean corpuscular volume (MCV) values. It is myoglobin) (Wells et al., 1980). This is a cold areas, thus lacking this phylogenetic the hematocrit value. Hematopoiesis is also possible to observe a higher value of this phylogenetic adaptability of fish species living in adaptation. accelerated in cases of hyperthermia as a result index at low temperatures (6°C MCV = cold waters, which have a high level of nitric of decreasing the erythrocyte-carrying capacity 76.33±5.779 fl), respectively a lower value in (Root effect), resulting the increase of case of high temperatures (31°C MCV = A B hematocrit. The mechanism consists in 38.66±2.109 fl). The average value of mean activating circulating stress hormones corpuscular volume recorded at 18 oC (MCV = (catecholamine) in hyperthermia and implicitly 60.90±1.077 fl) can be found between the in hypoxia (Lai et al., 2006), which accelerate highest and lowest values. hematopoietic processes. Mean corpuscular hemoglobin showed the Platelets (PLT) represent the only lowest value under control conditions (18oC hematological parameter that does not show MCH = 11.1±0.345 pg), value close to that any variation regardless of temperature resulting from hyperthermia (31oC MCH = 3 o intensity (PLT = 20.55±0.402 x 10 /μL - 6 C; 11.56±1.522 pg). Under hypothermia, the mean PLT = 20.30±0.434 x 103/μL - 18oC; PLT = corpuscular hemoglobin was higher (6oC MCH C D 3 20.30±0.636 x 10 /ΜL - 31 °C). = 17.86±1.868 pg). The erythrocyte count variation, as can be Mean corpuscular hemoglobin concentration seen in Figure 1, also led to the change of the showed the following mean values: 6°C - erythrocyte indices. Thus, the gradual increase MCHC = 24.30±2.503 g/dL; 18°C - MCHC = in the number of erythrocytes is correlated 31.30±0.454 g/dL; 31°C - MCHC = (according to the calculation formula) with a 22.57±1.176 g/dL.

E F

G H

37 MONO = 3%; BAS = 7%. The values for the addition, in the case of hypothermia, glycaemia, where significant differences (P the three experimental groups have been leucocyte formula at 31oC are as follows: NEU = lymphocytosis is observed, the percentage <0.05) were recorded between the three groups: provided with the same fodder conditions. Fish 42%; EOS = 17%; LYM = 6%; MONO = 15%; exceeding the maximum threshold of 40% 6oC BG = 34.33±3.97 mg/dL; 18oC BG = are poikilothermic organisms, so under BAS = 20%. (LYM = 80%, in this case). 23.00±0.50 mg/dL; 31oC BG = 27.66±2.15 conditions of temperature that do not fall The low percentage of neutrophils under The data presented in Table 1 (including the F mg/dL. Other experiments (Suljević et al., within the limits of their biological comfort, hypothermia (6°C) can be correlated with the values) reveal extremely significant differences 2015) also demonstrate changes in blood they eat small amounts of food or can even mean values obtained in the hematological (p <0.0001) depending on the temperature for glucose levels under thermic stress conditions. refuse feed altogether. Ingestion and food profile (low RBC, low Ht) and an inefficient most determined blood biochemical As for the mineral profile, the total calcium intake are limited in high temperature hematopoiesis at low temperatures (Kulkeaw et parameters, except for total calcium (TC) level does not differ significantly regardless of conditions involving hypoxia (Saravanan et al., al., 2010). Moreover, from the leukocyte where the difference among lots was the water temperature (6°C TC = 4.84±0.17 2013). In fact, extreme temperatures have a formula point of view, the extreme values were insignificant (p> 0.05). Also, a significant mg/dL, 18°C TC = 4.90±0.28 mg/dL, 31°C TC negative influence on metabolic processes, obtained under conditions of hypothermia, difference (p <0.05) was recorded in the case of = 4.62±0.26 mg/DL), as confirmed by other which is why ALP has declined, but without which means that the physiological status of glucose (BG). studies (Grigg, 1969; Cataldi et al., 1998). The reaching values that fall within pathological the Brown bullhead is strongly influenced. In mean values of the other mineral parameters spectrum. Being involved in physiological show extremely significant differences between calcium absorption processes (Villanueva et al., Table 1. The mean values, multiple comparisons (ANOVA test) and the statistical significance for the metabolic profiles and the three groups. 1997), correlations can be made between total the oxidative stress in situations of thermal stress in Brown bullhead (Ameiurus nebulosus) The values for iron (Fe) (6°C Fe = 283.18±0.23 calcium (TC) and alkaline phosphatase (ALP)

Parameter Abbr. MU LT* NT* HT* F* Sign.P μg/dL; 18°C Fe = 97.27±10.74 μg/dL; 31°C Fe values in thermal stress situations. Proteic Profile = 230.23±30.53 μg/dL) may be correlated with Antagonist to ALP values, gamma- Total Protein TP g/dL 0.98±0.04 3.30±0.33 2.36±0.38 15.58 <0.0001 blood hemoglobin level (6°C Hg = 10.2±0.388 glutamyltransferase (GGT) presents elevated Albumin ALB g/dL 2.34±0.05 1.30±0.13 1.53±0.21 13.55 <0.0001 o o Globulin GLOB g/dL 0.25±0.01 3.20±0.19 2.76±0.24 78.39 <0.0001 g/dL, 18 C Hg = 5.5±0.196 g/dL, 31 C Hg = values in thermal stress situations, suggesting Blood Urea Nitrogen BUN mg/dL 10.33±0.52 4.00±0.30 7.74±0.41 56.51 <0.0001 7.66±0.638 g/dL), demonstrating that both low poor metabolic activity of the liver and a Creatine CREAT mg/dL 0.79±0.05 0.27±0.01 0.71±0.07 27.36 <0.0001 and high water temperatures lead to decrease in bile secretion. In the case of alanine Lipid Profile Cholesterol CHOL mg/dL 64.33±2.75 105.00±2.29 191.33±14.15 59.25 <0.0001 disturbances of the respiratory function because aminotransferase (ALT), the values are Triglyceride TGC mg/dL 100.33±2.51 329.00±9.69 282.33±25.47 58.47 <0.0001 of peripheral vasoconstriction (low elevated only under high temperature Carbohydrate Profile temperature) or lack of dissolved oxygen (high conditions (6oC ALT = 14.66±0.57 U/L, 18oC Blood Glucose BG mg/dL 34.33±3.97 23.00±0.50 27.66±2.15 4.70 <0.05 o Mineral Profile temperatures). ALT = 14.00±0.36 U/L, 31 C ALT = Total Calcium TC mg/dL 4.84±0.17 4.90±0.28 4.62±0.26 0.36 ns At an enzymatic level, significant differences 39.10±4.17 U/L). Serum amylase (AM EP) Phosphorus P mg/dL 2.53±0.12 3.12±0.34 9.05±1.06 31.04 <0.0001 were obtained between the three groups. The also showed elevated levels of thermal stress Blood Iron Fe µg/dL 283.18±0.23 97.27±10.74 230.23±30.53 17.46 <0.0001 most dramatic differences were recorded in due to a pancreatic dysfunction or a possible Sodium Na mmol/L 129.00±1.75 111.70±2.02 121.33±1.38 25.04 <0.0001 o Potassium K mmol/L 3.50±0.17 2.21±0.21 2.44±0.08 17.03 <0.0001 aspartate aminotransferase (6 C AST = renal insufficiency due to extreme Enzyme Profile 1916.66±25.58 U/L; 18oC AST = 268.00±3.51 temperatures. Danilenko et al. (1998), confirms Alkaline Phosphatase ALP UL 65.33±3.02 155.00±3.13 121.66±6.93 91.9 <0.0001 U/L; 31oC AST = 1448.00±147.8 U/L), where changes in lactate dehydrogenase (LDH) values Gamma-glutamyl transpeptidase GGT UL 16.80±1.1 2.00±0.21 18.66±0.48 149.1 <0.0001 Aspartate aminotransferase AST UL 1916.66±25.58 268.00±3.51 1448.00±147.8 96.13 <0.0001 in conditions of hypothermia, values in fish depending on temperature, the same fact Alanine aminotransferase ALT UL 14.66±0.57 14.00±0.36 39.10±4.17 34.33 <0.0001 approximately seven times higher appeared, being confirmed by the results of our study Serum Amylase AM EP UL 135.66±9.04 44.00±2.49 84.00±2.70 66.5 <0.0001 and in hyperthermia conditions the standard (6oC LDH = 1467.00±17.02 U/L; 18oC LDH = Lactate Dehydrogenase LDH UL 1467.00±17.02 282.00±3.06 502.66±24.58 1319 <0.0001 o o Lipase LIP UL 295.66±4.73 55.00±3.14 124.00±12.95 230.4 <0.0001 value (at 18 C) was approximately five times 282.00±3.06 U/L; 31 C LDH = 502.66±24.58 Creatine Phosphokinase CPK UL 12024.33±265.4 17620.00±190.1 9523.00±393.7 197.1 <0.0001 higher. These values can conclude the U/L). Comparing the mean LDH values with Total Bilirubin TB UL 0.55±0.02 0.08±0.01 0.04±0.01 295.5 <0.0001 occurrence of tissue lesions (Preston et al., the value obtained at 18°C in both hypothermia Oxidative Stress Superoxide Dismutase SOD U/gHGB 823.51±43.71 238.53±34.12 706.63±47.3 54.11 <0.0001 2016), AST being an enzyme found in several and hyperthermia, these are higher, resulting in *LT – lower temperature (6oC); NT – normal temperature (18oC); HT – high temperature (31oC); F – F statistic types of tissue (myocardium, liver, skeletal extremely significant differences between the muscle, pancreas, kidney). three groups (F = 1319; P <0.0001). LDH Protein profile parameters can vary depending proteins in the blood does not necessarily In situations of thermal stress, alkaline elevations may appear due to hypoxia on several factors: species, sex, age, water represent a state of stress, this fact being phosphatase (ALP) showed lower values (Panepucci et al., 2000) in the case of high temperature, feed (Patriche et al., 2009). So, in attributed to the ratio of albumin to globulin, compared to those obtained at 18oC (ALP = water temperatures. our case, the variation of protein parameters which should not fall below 0.3. Values 155.00±3.13 U/L), suggesting a slight was influenced only by water temperature, as obtained show the following albumin / globulin malnutrition of specimens exposed to low CONCLUSIONS o o the brown bullheads were of the same age and ratios: 6 C ALB / GLOB = 9.35; 18 C ALB / temperature (6oC ALP = 65.33±3.02 U/L), o received the same type of feed. The same GLOB = 0.406; 31 C ALB / GLOB = 0.554. respectively to high temperature (31°C ALP = The results of our experiment indicate the fact authors (Patriche et al., 2011) consider that the According to the same authors, a stress 121.66±6.93 U/L). This slight malnutrition is that Brown bullhead has great plasticity and difference between the groups of circulating indicator valuable biomarker is the level of not the result of inappropriate feeding, because adaptability regarding environmental

38 MONO = 3%; BAS = 7%. The values for the addition, in the case of hypothermia, glycaemia, where significant differences (P the three experimental groups have been leucocyte formula at 31oC are as follows: NEU = lymphocytosis is observed, the percentage <0.05) were recorded between the three groups: provided with the same fodder conditions. Fish 42%; EOS = 17%; LYM = 6%; MONO = 15%; exceeding the maximum threshold of 40% 6oC BG = 34.33±3.97 mg/dL; 18oC BG = are poikilothermic organisms, so under BAS = 20%. (LYM = 80%, in this case). 23.00±0.50 mg/dL; 31oC BG = 27.66±2.15 conditions of temperature that do not fall The low percentage of neutrophils under The data presented in Table 1 (including the F mg/dL. Other experiments (Suljević et al., within the limits of their biological comfort, hypothermia (6°C) can be correlated with the values) reveal extremely significant differences 2015) also demonstrate changes in blood they eat small amounts of food or can even mean values obtained in the hematological (p <0.0001) depending on the temperature for glucose levels under thermic stress conditions. refuse feed altogether. Ingestion and food profile (low RBC, low Ht) and an inefficient most determined blood biochemical As for the mineral profile, the total calcium intake are limited in high temperature hematopoiesis at low temperatures (Kulkeaw et parameters, except for total calcium (TC) level does not differ significantly regardless of conditions involving hypoxia (Saravanan et al., al., 2010). Moreover, from the leukocyte where the difference among lots was the water temperature (6°C TC = 4.84±0.17 2013). In fact, extreme temperatures have a formula point of view, the extreme values were insignificant (p> 0.05). Also, a significant mg/dL, 18°C TC = 4.90±0.28 mg/dL, 31°C TC negative influence on metabolic processes, obtained under conditions of hypothermia, difference (p <0.05) was recorded in the case of = 4.62±0.26 mg/DL), as confirmed by other which is why ALP has declined, but without which means that the physiological status of glucose (BG). studies (Grigg, 1969; Cataldi et al., 1998). The reaching values that fall within pathological the Brown bullhead is strongly influenced. In mean values of the other mineral parameters spectrum. Being involved in physiological show extremely significant differences between calcium absorption processes (Villanueva et al., Table 1. The mean values, multiple comparisons (ANOVA test) and the statistical significance for the metabolic profiles and the three groups. 1997), correlations can be made between total the oxidative stress in situations of thermal stress in Brown bullhead (Ameiurus nebulosus) The values for iron (Fe) (6°C Fe = 283.18±0.23 calcium (TC) and alkaline phosphatase (ALP)

39 conditions. Hematologic and metabolic profiles Fobes M.C., 2013. Controlled spawning of White haematological profile of juvenile Cyprinus carpio Nebulosus Nebulosus (LeSueur), in Cayuga Lake, show changes when thermal stress is induced. Catfish, Ictalurus catus, and Brown Bullhead Catfish, varieties. Scientific Papers. Series D. Animal New York. Transaction of the American Fisheries Ameiurus nebulosus, using carp pituitary extract and Science, Vol. LVIII, p. 209-212. Society, 69(1): p. 205-209. No pathologic state was recorded during the LHRHa. Master of Science Thesis, Auburn Marković G.S., Ćirković M.A., Maletin S.A., 2012. The Rowan M.W., 2007. Use of blood parameters as experiment, but changes in behaviour were University, Alabama, USA. role of allochthonous (non-native) fish species in biomarkers in brown bullhead (Ameiurus nebulosus) o observed. Both for low temperature (6 C) and Frank K.D., 2015. Ecology of Center City, Philadelphia. Serbian aquaculture. Journal of Central European from Lake Erie tributaries and Cape Cod ponds. high temperature (31oC) fish reacted as a Chapter 21: Brown Bullhead (Ameiurus nebulosus). Agriculture, 13(3): p. 539-544. Dissertation Thesis, School of the Ohio State lethargic condition set off. We assume that Publisher: Filter Square Press, p. 215-227. Martínez-Porchas M., Martínez-Córdova L.F., Ramos- University, Ohio State University, USA. Ghergariu S., Pop A., Kadar L., Spînu M., 1999. The Enriquez R., 2009. Cortisol and glucose: Reliable Rummer J.L., Brauner C.J., 2015. Root effect Brown bullhead has a more complex Veterinary Clinical Laboratory Manual. All indicators of fish stress? Pan-American Journal of haemoglobins in fish may greatly enhance general mechanism in terms of thermal stress states. Publishing House, Bucharest. Aquatic Sciences, 4(2): p. 158-178. oxygen delivery relative to other vertebrates. PLoS Gillooly J.F., Zenil-Ferguson R., 2014. Vertebrate blood Moldowan P.D., Keevil M.G., Kell S., Brooks R.J., ONE, 10(10):e0139477. REFERENCES cell volume increase with temperature: implication Litzgus J.D., 2015. Anti-predator defenses of Brown DOI:10.1371/journal.pone.0139477. for aerobic activity. PeerJ, DOI 10.7717/peerj.346. Bullheads (Ameiurus nebulosus) and interactions Saravanan S., Geurden I., Figueiredo-Silva A.C., Bănărescu P., 1964. Fauna of the România People's Grigg G.C., 1969. Temperature-induced changes in the with Snapping Turtles (Chelydra serpentina). The Nusantoro S., Kaushik S., Verreth J., Schrama J.W., Republic. Pisces-Osteichthyes, vol. XIII, p. 552-555. oxygen equilibrium curve of the blood of the Brown Canadian Field-Naturalist, 129(2): p. 189-193. 2013. Oxygen consumption constrains food intake in Barton B.A., 2002. Stress in fishes: a diversity of Bullhead, Ictalurus nebulosus. Comparative Panepucci L., Fernandes M.N., Sanches J.R., Rantin fish fed diets varying in essential amino acids responses with particular reference to changes in Biochemistry and Physiology, 28: p. 1203-1223. F.T., 2000. Changes in lactate dehydrogenase and composition. PLoS ONE 8(8): e72757. doi: 10. circulating corticosteroids. Integrative and Hattingh J., 1977. Blood sugar as an indicator of stress in malate dehydrogenase activities during hypoxia and 1371/journal.pone.0072757. Comparative Biology, 42(3): p. 517-525. the freshwater fish, Labeo capensis (Smith). Journal after temperature acclimation in the armored fish, Scott W.B., Crossman E.J., 1973. Freshwater Fishes of Baumann P.C., LeBlanc D.R., Blazer V.S., Meier J.R., of Fish Biology, 10(2): p. 191-195. Rhinelepis strigosa (Siluriformes, Loricariidae). Canada. Bulletin 184. Fisheries Research Board of Hurley S.T., Kiryu Y., 2008. Prelevance of tumors in Holčik J., 1991. 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Determination Simide R., Richard S., Prévot-D'Alvise N., Miard T., blood parameters of common carp (Cyprinus carpio). reference to teleost fish. Symposia of the Society for of some normal parameters in juvenile Sevruga Gaillard S., 2016. Assessment of the accuracy of 2nd International Conference on Environmental Experimental Biology, 41: p. 67-93. sturgeons Acipenser stellatus (Pallas, 1771). Archiva physiological blood indicators for the evaluation of Science and Technology, IPCBEE 6(2): p. 52-55. Kapusta A., Morzuch J., Partyka K., Bogacka-Kapusta Zootechnica, 14(1): p. 49-54. stress, health status and welfare in Siberian sturgeon Cataldi E., Di Marco P., Mandich A., Cataudella S., E., 2010. First record of brown bullhead, Ameiurus Peck M.A., Buckley L.J., Bengtson D.A., 2005. Effects (Acipenser baerii) subject to chronic heat stress and 1998. 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40 conditions. Hematologic and metabolic profiles Fobes M.C., 2013. Controlled spawning of White haematological profile of juvenile Cyprinus carpio Nebulosus Nebulosus (LeSueur), in Cayuga Lake, show changes when thermal stress is induced. Catfish, Ictalurus catus, and Brown Bullhead Catfish, varieties. Scientific Papers. Series D. Animal New York. Transaction of the American Fisheries Ameiurus nebulosus, using carp pituitary extract and Science, Vol. LVIII, p. 209-212. Society, 69(1): p. 205-209. No pathologic state was recorded during the LHRHa. Master of Science Thesis, Auburn Marković G.S., Ćirković M.A., Maletin S.A., 2012. The Rowan M.W., 2007. Use of blood parameters as experiment, but changes in behaviour were University, Alabama, USA. role of allochthonous (non-native) fish species in biomarkers in brown bullhead (Ameiurus nebulosus) o observed. Both for low temperature (6 C) and Frank K.D., 2015. Ecology of Center City, Philadelphia. Serbian aquaculture. Journal of Central European from Lake Erie tributaries and Cape Cod ponds. high temperature (31oC) fish reacted as a Chapter 21: Brown Bullhead (Ameiurus nebulosus). Agriculture, 13(3): p. 539-544. Dissertation Thesis, School of the Ohio State lethargic condition set off. We assume that Publisher: Filter Square Press, p. 215-227. Martínez-Porchas M., Martínez-Córdova L.F., Ramos- University, Ohio State University, USA. Ghergariu S., Pop A., Kadar L., Spînu M., 1999. The Enriquez R., 2009. Cortisol and glucose: Reliable Rummer J.L., Brauner C.J., 2015. Root effect Brown bullhead has a more complex Veterinary Clinical Laboratory Manual. All indicators of fish stress? Pan-American Journal of haemoglobins in fish may greatly enhance general mechanism in terms of thermal stress states. Publishing House, Bucharest. Aquatic Sciences, 4(2): p. 158-178. oxygen delivery relative to other vertebrates. PLoS Gillooly J.F., Zenil-Ferguson R., 2014. Vertebrate blood Moldowan P.D., Keevil M.G., Kell S., Brooks R.J., ONE, 10(10):e0139477. REFERENCES cell volume increase with temperature: implication Litzgus J.D., 2015. Anti-predator defenses of Brown DOI:10.1371/journal.pone.0139477. for aerobic activity. PeerJ, DOI 10.7717/peerj.346. Bullheads (Ameiurus nebulosus) and interactions Saravanan S., Geurden I., Figueiredo-Silva A.C., Bănărescu P., 1964. Fauna of the România People's Grigg G.C., 1969. Temperature-induced changes in the with Snapping Turtles (Chelydra serpentina). The Nusantoro S., Kaushik S., Verreth J., Schrama J.W., Republic. Pisces-Osteichthyes, vol. XIII, p. 552-555. oxygen equilibrium curve of the blood of the Brown Canadian Field-Naturalist, 129(2): p. 189-193. 2013. Oxygen consumption constrains food intake in Barton B.A., 2002. Stress in fishes: a diversity of Bullhead, Ictalurus nebulosus. Comparative Panepucci L., Fernandes M.N., Sanches J.R., Rantin fish fed diets varying in essential amino acids responses with particular reference to changes in Biochemistry and Physiology, 28: p. 1203-1223. F.T., 2000. Changes in lactate dehydrogenase and composition. PLoS ONE 8(8): e72757. doi: 10. circulating corticosteroids. Integrative and Hattingh J., 1977. Blood sugar as an indicator of stress in malate dehydrogenase activities during hypoxia and 1371/journal.pone.0072757. Comparative Biology, 42(3): p. 517-525. the freshwater fish, Labeo capensis (Smith). Journal after temperature acclimation in the armored fish, Scott W.B., Crossman E.J., 1973. Freshwater Fishes of Baumann P.C., LeBlanc D.R., Blazer V.S., Meier J.R., of Fish Biology, 10(2): p. 191-195. Rhinelepis strigosa (Siluriformes, Loricariidae). Canada. Bulletin 184. Fisheries Research Board of Hurley S.T., Kiryu Y., 2008. Prelevance of tumors in Holčik J., 1991. Fish introduction in Europe with Revista Brasileira de Biologia, 60(2): p. 353-360. Canada, 966 p. Brown bullhead from three lakes in southeastern particular reference to its central and Eastern Part. Patriche T., Patriche N., Tenciu M., 2009. Cyprinids Sidell B.D., O'Brien K.M., 2006. When bad things Massachusetts, 2002. US Geological Survey Canadian Journal of Fisheries and Aquatic Sciences, total blood proteins determination. Lucrări Ştiinţifice happen to good fish: the loss of hemoglobin and Scientific Investigation Report 2008-5198, 43 p. 48(1): p. 13-23. Zootehnie şi Biotehnologii, UASVMB Timişoara, myoglobin expression in Antarctic icefishes. Journal Bozorgnia A., Hosseinifard M., Alimohammadi R., Johnson I.A., Dunn J., 1987. Temperature acclimation 42(2): p. 95-101. of Experimental Biology, 209: p. 1791-1802. 2011. Acute effects of different temperature in the and metabolism in ectotherms with particular Patriche T., Patriche N., Bocioc E., 2011. 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